Abstract This proposal describes a 2-year research plan focusing on the effect of Foxp3 methylation by PRMT5 on regulatory T cells (Tregs). Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by the development of multiple autoimmune disorders due to defective Tregs. Tregs limit excessive inflammation, especially those that are directed towards self antigens. IPEX is caused by mutations of the Foxp3 gene. However, the severity of IPEX does not always correlate with the level of Foxp3 protein expression, suggesting that IPEX can occur as a result of post- translational defects in Foxp3. Thus, it is important to investigate how Foxp3 function is regulated by post- translational mechanisms. The function of Foxp3 is influenced by post-translational acetylation and ubiquitination. In addition to these post-translational modifications, we hereby propose that methylation of Foxp3 is a critical regulator of Foxp3 function. Our preliminary data show that Foxp3 directly associates with and is methylated by Protein Arginine Methyltransferase 5 (PRMT5). Importantly, the conditional ablation of loxp-flanked PRMT5 (PRMT5- flox) specifically in Tregs using a Foxp3-induced Cre resulted in a severe and fatal scurfy-like autoimmune phenotype, characterized by progressive weight loss, dermatitis, and splenomegaly, with death occurring by 40 days after birth. Peripheral Tregs were almost completely absent in the spleen and significantly reduced in the lymph nodes of PRMT5-FOXP3-Cre mice, suggesting that Treg development and/or stability was severely diminished. Lymph node Tregs from PRMT5-FOXP3-Cre mice also showed diminished suppressive function. Based on these preliminary data, we hypothesize that methylation of Foxp3 by PRMT5 is required for normal Treg development and function. We will test this hypothesis in 2 Aims. In Aim 1, we will fully characterize the role of PRMT5 in thymic and peripheral Treg development and test whether PRMT5 is required for mature Tregs to maintain their Treg phenotype and to carry out their suppressive function. In Aim 2, we will test whether PRMT5 methylates Foxp3 in Tregs and identify the amino acid residues that are methylated in Foxp3 by PRMT5. We believe that the experiments outlined in this proposal will yield insight into a novel mechanism of Foxp3 regulation by post- translational methylation. The research team is comprised of Drs. Yasuhiro Nagai (expert in biochemistry and human T cell manipulation), Mark Greene (expert in biochemistry and structural biology), and Taku Kambayashi (expert in cell signaling and regulatory T cell biology). The synergy of the investigators' expertise will increase the likelihood of the success of the project. Completion of these aims will also allow us to rigorously determine the role of Foxp3 methylation by PRMT5 in Treg function and development, and will provide a novel strategy for diagnostics in IPEX and a new strategy for therapy of IPEX patients.